Miroslaw Gilski

Crystal structure of a monomeric retroviral protease solved by protein folding game players

Following the failure of a wide range of attempts to solve the crystal structure of M-PMV retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs.

Stereochemical restraints revisited: how accurate are refinement targets and how much should protein structures be allowed to deviate from them?

The Protein Data Bank and Cambridge Structural Database were analyzed with the aim of verifying whether the restraints that are most commonly used for protein structure refinement are still appropriate 15 years after their introduction. From an analysis of selected main-chain parameters in well ordered fragments of ten highest resolution protein structures, it was concluded that some of the currently used geometrical target values should be adjusted somewhat (the C-N bond and the N-C(alpha)-C angle) or applied with less emphasis (peptide planarity). It was also found that the weighting of stereochemical information in medium-resolution refinements is often overemphasized at the cost of the experimental information in the diffraction data. A correctly set balance will be reflected in root-mean-square deviations from ideal bond lengths in the range 0.015-0.020 A for structures refined to R factors of 0.15-0.20. At ultrahigh resolution, however, the diffraction terms should be allowed to dominate, with even higher acceptable deviations from idealized standards in the well defined fragments of the protein. It is postulated that modern refinement programs should accommodate variable restraint weights that are dependent on the occupancies and B factors of the atoms involved.

Mechanochemical Encapsulation of Fullerenes in Peptidic Containers Prepared by Dynamic Chiral Self‐Sorting and Self‐Assembly

Molecular capsules composed of amino acid or peptide derivatives connected to resorcin[4]arene scaffolds through acylhydrazone linkers have been synthesized using dynamic covalent chemistry (DCC) and hydrogen-bond-based self-assembly. The dynamic character of the linkers and the preference of the peptides towards self-assembly into β-barrel-type motifs lead to the spontaneous amplification of formation of homochiral capsules from mixtures of different substrates. The capsules have cavities of around 800 Å(3) and exhibit good kinetic stability. Although they retain their dynamic character, which allows processes such as chiral self-sorting and chiral self-assembly to operate with high fidelity, guest complexation is hindered in solution. However, the quantitative complexation of even very large guests, such as fullerene C60 or C70 , is possible through the utilization of reversible covalent bonds or the application of mechanochemical methods. The NMR spectra show the influence of the chiral environment on the symmetry of the fullerene molecules, which results in the differentiation of diastereotopic carbon atoms for C70 , and the X-ray structures provide unique information on the modes of peptide-fullerene interactions.

High-resolution structure of a retroviral protease folded as a monomer.

The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs.

Structure of γ-conglutin: insight into the quaternary structure of 7S basic globulins from legumes

γ-Conglutin from lupin seeds is an unusual 7S basic globulin protein. It is capable of reducing glycaemia in mammals, but the structural basis of this activity is not known. γ-Conglutin shares a high level of structural homology with glycoside hydrolase inhibitor proteins, although it lacks any kind of inhibitory activity against plant cell-wall degradation enzymes. In addition, γ-conglutin displays a less pronounced structural similarity to pepsin-like aspartic proteases, but it is proteolytically dysfunctional. Only one structural study of a legume 7S basic globulin, that isolated from soybean, has been reported to date. The quaternary assembly of soybean 7S basic globulin (Bg7S) is arranged as a cruciform-shaped tetramer comprised of two superposed dimers. Here, the crystal structure of γ-conglutin isolated from Lupinus angustifolius seeds (LangC) is presented. The polypeptide chain of LangC is post-translationally cleaved into α and β subunits but retains its covalent integrity owing to a disulfide bridge. The protomers of LangC undergo an intricate quaternary assembly, resulting in a ring-like hexamer with noncrystallographic D3 symmetry. The twofold-related dimers are similar to those in Bg7S but their assembly is different as a consequence of mutations in a β-strand that is involved in intermolecular β-sheet formation in γ-conglutin. Structural elucidation of γ-conglutin will help to explain its physiological role, especially in the evolutionary context, and will guide further research into the hypoglycaemic activity of this protein in humans, with potential consequences for novel antidiabetic therapies.

Crystal Structure of 2′-Deoxycytidine Hemidihydrogenphosphate Reveals C+·C Base Pairs and Tight, Hydrogen-Bonded (H2PO4 −)∞ Columns (1)

Abstract 2′-Deoxycytidine hemidihydrogenphosphate has been crystallized in the hexagonal space group P62 with α=25.839(3), c = 12.529(1) A. The structure has been solved using the Patterson search method. The asymmetric unit contains two protonated, base-paired 2′-deoxycytidine dimers and two H2PO4 − anions. The C+·C base pairs are composed of a protonated and a neutral species each and are triple H-bonded, the central N(3)…N(3) bonds being 2.850(7) and 2.884(5) A. The conformations of the four nucleosides fall in the same category (sugar puckers 2·-endo, glycosidic links anti) but in one of them the glycosidic torsion angle is quite low with consequences in other geometrical parameters. The H2PO4 − anions are located on twofold axes and form two types of tight columns with P…P separations about 4.18 A The neighboring units along a column are linked via two very short O…H…O hydrogen bonds (O…O about 2.49 A) leading to effective equalization of the P-O bonds. The base pairs of the two dC+·dC cations are co...

Numerology versus reality: a voice in a recent dispute

Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University and Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland, Synchrotron Radiation Research Section, Macromolecular Crystallography Laboratory, NCI, Argonne National Laboratory, Biosciences Division, Building 202, Argonne, IL 60439, USA, and Protein Structure Section, Macromolecular Crystallography Laboratory, NCI at Frederick, Frederick, MD 21702, USA

Atomic resolution structure of a chimeric DNA-RNA Z-type duplex in complex with Ba(2+) ions: a case of complicated multi-domain twinning.

The self-complementary dCrGdCrGdCrG hexanucleotide, in which not only the pyrimidine/purine bases but also the ribo/deoxy sugars alternate along the sequence, was crystallized in the presence of barium cations in the form of a left-handed Z-type duplex. The asymmetric unit of the P21 crystal with a pseudohexagonal lattice contains four chimeric duplexes and 16 partial Ba(2+) sites. The chimeric (DNA-RNA)2 duplexes have novel patterns of hydration and exhibit a high degree of discrete conformational disorder of their sugar-phosphate backbones, which can at least partly be correlated with the fractional occupancies of the barium ions. The crystals of the DNA-RNA chimeric duplex in complex with Ba(2+) ions and also with Sr(2+) ions exhibit complicated twinning, which in combination with structural pseudosymmetry made structure determination difficult. The structure could be successfully solved by molecular replacement in space groups P1 and P21 but not in orthorhombic or higher symmetry and, after scrupulous twinning and packing analysis, was refined in space group P21 to an R and Rfree of 11.36 and 16.91%, respectively, using data extending to 1.09 Å resolution. With the crystal structure having monoclinic symmetry, the sixfold crystal twinning is a combination of threefold and twofold rotations. The paper describes the practical aspects of dealing with cases of complicated twinning and pseudosymmetry, and compares the available software tools for the refinement and analysis of such cases.

Experiences with applications of macromolecular tools in supramolecular crystallography

Supramolecular structures, with ever increasing size ranging from a few up to tens of nanometres, represent an intermediate stage between small molecules and biological macromolecules. Many crystal structures of these large supramolecular assemblies have been solved using dual space algorithms. However, supramolecular assemblies with a capsular shape present a particular challenge for crystallography, especially when they are chiral and composed of only light atoms. In this paper, we show that the application of “routine” macromolecular tools may be of great help in solving the crystal structures of supramolecular assemblies that are otherwise refractory to the routine methods of small molecule crystallography. Specifically, we have applied the method of molecular replacement as implemented in PHASER in order to solve the crystal structure of a chiral organic capsule, which could not be determined using direct or dual space methods. By utilizing various models consisting of well-defined supramolecular “bricks” or modelled structures, we show how model size (fraction of the asymmetric unit) and quality (root mean square deviation from target) influence the success rate of medium sized non-protein structures. The results indicate that supramolecular structures, that are still “small molecules” for macromolecular standards, can be successfully solved using even very small models, down to 25% by weight of the contents of the asymmetric unit.

Contribution of Leptosphaeria species ascospores to autumn asthma in areas of oilseed rape production

BACKGROUND An increase in the number of hospital admissions from September to November in the northern hemisphere has been frequently reported. At this time, some species of fungal genus Leptosphaeria produce numerous ascospores, which are easily airborne. However, we lack knowledge about whether Leptosphaeria produces allergenic proteins. OBJECTIVE To evaluate the potential of Leptosphaeria ascospores to contribute to autumn asthma. METHODS Detailed bioinformatic analysis of proteins produced by Leptosphaeria maculans available in databases was performed and the data compared with allergens found in other airborne fungi. The concentrations of Leptosphaeria ascospores detected at 2 sites were compared to these obtained in other environments worldwide. RESULTS We found that Leptosphaeria species produce proteins with a high identity to commonly known aeroallergens of several well-characterized molds. The level of amino acid identity significantly exceeded the allergen identity thresholds recommended by the Food and Agricultural Organization/World Health Organization (35%), which indicates allergenic properties of L maculans and ensures the same properties in the other Leptosphaeria species. CONCLUSION High concentrations of Leptosphaeria species ascospores in the autumn and postulated allergenicity of their proteins strongly suggest that this genus contributes to worldwide reported autumn asthma. The finding opens the question of allergenicity of the other never studied fungal species present in aeroplankton.